US5028537A - Novel antifungal substances and process for their production - Google Patents

Novel antifungal substances and process for their production Download PDF

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Publication number
US5028537A
US5028537A US07/202,612 US20261288A US5028537A US 5028537 A US5028537 A US 5028537A US 20261288 A US20261288 A US 20261288A US 5028537 A US5028537 A US 5028537A
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compounds
compound
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US07/202,612
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Richard W. Burg
Otto D. Hensens
Jerrold M. Liesch
Sebastian Hernandez
Lucille J. Cole
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Merck and Co Inc
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Merck and Co Inc
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Priority claimed from US06/593,448 external-priority patent/US4575500A/en
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Priority to US07/202,612 priority Critical patent/US5028537A/en
Priority to GB8912558A priority patent/GB2219296A/en
Priority to US07/660,201 priority patent/US5130445A/en
Assigned to MERCK & CO., INC., A CORP. OF NEW JERSEY reassignment MERCK & CO., INC., A CORP. OF NEW JERSEY ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: COLE, LUCILLE J., HERNANDEZ, SEBASTIAN, BURG, RICHARD W., HENSENS, OTTO D., LIESCH, JERROLD M.
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
    • A01N57/12—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing acyclic or cycloaliphatic radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/117—Esters of phosphoric acids with cycloaliphatic alcohols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces
    • C12R2001/55—Streptomyces hygroscopicus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/886—Streptomyces
    • Y10S435/898—Streptomyces hygroscopicus

Definitions

  • This invention is concerned with novel organic chemical compounds.
  • it is concerned with phosphate esters which are produced by the fermentation of a nutrient medium with a strain of the microorganism Streptomyces hygroscopicus MA-5000.
  • it is an object of this invention to provide for such novel compounds, and a method for preparing such products microbiologically.
  • These substances have antifungal activity, and it is, thus, an additional object of this invention to provide for novel antifungal compositions containing the disclosed compounds. Further objects of this invention will become apparent from the following description of this invention.
  • novel substances which are prepared by growing under controlled conditions, a previously undescribed strain of a microorganism.
  • novel compounds are produced by Streptomyces hygroscopicus MA-5000.
  • the compounds are obtained by fermentation and recovered in substantially pure form as described herein.
  • FIG. 1 is a nuclear magnetic resonance spectrum for Compound I.
  • FIG. 2 is a nuclear magnetic resonance spectrum for Compound II.
  • FIG. 3 is a nuclear magnetic resonance spectrum for Compound III.
  • FIG. 4 is a nuclear magnetic resonance spectrum for Compound IV.
  • the microorganism capable of producing these compounds is of a new strain of the microorganism Streptomyces hygroscopicus.
  • One such culture, isolated from soil, is designated MA-5000 in the culture collection of Merck & Co., Inc., Rahway, N.J.
  • a sample of this culture, capable of producing the herein described compound, has been deposited, without restriction as to availability, in the permanent culture collection of the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Md. 20852, and has been assigned the accession number ATCC 39476.
  • Streptomyces hygroscopicus MA-5000 The morphological and cultural characteristics of Streptomyces hygroscopicus MA-5000 are set forth below:
  • Morphology Sporophores form compact spirals along aerial hyphae. As culture ages, spore chains coalesce to form dark moist areas. Spore surface by electron microscope (TEM) shows a smooth surface on some spores and a rough to warty surface on others.
  • TEM electron microscope
  • A Dark gray mixed with white and light gray, becoming black and moist with age.
  • A Light gray mixed with white and edged with dark gray, some areas becoming moist and black.
  • A Light gray mixed with white and yellowish white. Droplets of golden yellow exudate are present.
  • ISP Medium 4 Inorganic salts-starch agar
  • A Dark gray mixed with light gray and white, becoming black and moist.
  • A Dark gray becoming black and moist with age. Droplets of golden yellow exudate also present.
  • the above description is illustrative of a strain of Streptomyces hygroscopicus MA-5000 which can be employed in the production of the instant compound.
  • the present invention also embraces mutants of the above described microorganism. For example, those mutants which are obtained by natural selection or those produced by mutating agents including ionizing radiation such as ultraviolet irradiation, or chemical mutagens such as nitrosoguanidine or the like treatments are also included within the ambit of this invention.
  • the instant compounds are produced during the aerobic fermentation of suitable aqueous nutrient media under conditions described hereinafter, with a producing strain of Streptomyces hygroscopicus MA-5000.
  • Aqueous media such as those used for the production of many antibiotic substances are suitable for use in this process for the production of this macrocyclic compound.
  • Such nutrient media contain sources of carbon and nitrogen assimilable by the microorganism and generally low levels of inorganic salts.
  • the fermentation media may contain traces of metals necessary for the growth of the microorganisms, and production of the desired compound. These are usually present in sufficient concentrations in the complex sources of carbon and nitrogen, which may be used as nutrient sources, but can, of course, be added separately to the medium if desired.
  • carbohydrates such as sugars, for example dextrose, sucrose, maltose, lactose, dextran, cerelose, corn meal, oat flour, and the like, and starches are suitable sources of assimilable carbon in the nutrient media.
  • the exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between 0.5 and 5% by weight of the medium is satisfactory.
  • These carbon sources can be used individually or several such carbon sources may be combined in the same medium.
  • Various nitrogen sources such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, corn meal, oat flour, soybean meal, caesin hydrolysates, yeast extracts, corn steep liquors, distillers solubles, cottonseed meal, meat extract and the like, are readily assimilable by Streptomyces hygroscopicus MA 5000 in the production of the instant compound.
  • the various sources of nitrogen can be used alone or in combination in amounts ranging from 0.2 to 6% by weight of the medium.
  • the customary salts capable of yielding sodium, potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride, carbonate, and like ions. Also included are trace metals such as cobalt, manganese, and the like. For the best production of the instant compound, the addition of calcium carbonate to the production medium is most preferred.
  • the fermentation employing Streptomyces hygroscopicus MA 5000 can be conducted at temperatures ranging from about 20° C. to about 40° C. For optimum results, it is most convenient to conduct these fermentations at a temperature in the range of from about 24° C. to about 30° C. Temperatures of about 27°-28° C. are most preferred.
  • the pH of the nutrient medium suitable for producing the instant compounds can vary from about 5.0 to 8.5 with a preferred range of from about 6.0 to 7.5.
  • Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in a flask employing known sterile techniques, inoculating the flask with either spores or vegetative cellular growth of Streptomyces hygroscopicus MA-5000 loosely stoppering the flask with cotton and permitting the fermentation to proceed in a constant room temperature of about 28° C. on a rotary shaker at from 95 to 300 rpm for about 2 to 10 days.
  • suitable tanks provided with an agitator and a means of aerating the fermentation medium.
  • the nutrient medium is made up in the tank and after sterilization is inoculated with a source of vegetative cellular growth of Streptomyces hygroscopicus MA 5000.
  • the fermentation is allowed to continue for from 1 to 8 days while agitating and/or aerating the nutrient medium at a temperature in the range of from about 24° to 37° C.
  • the degree of aeration is dependent upon several factors such as the size of the fermenter, agitation speed, and the like.
  • the larger scale fermentations are agitated at about 95 to 300 RPM and about 2 to 20 cubic feet per minute (CFM) of air.
  • novel compound of this invention is found primarily in the mycelium on termination of the Streptomyces hygroscopicus MA-5000 fermentation and may be removed and separated therefrom as described below.
  • the separation of the novel compound from the whole fermentation broth and the recovery of said compounds is carried out by solvent extraction and application of chromatographic fractionations with various chromatographic techniques and solvent systems.
  • the instant compound has slight solubility in water, but is soluble in organic solvents. This property may be conveniently employed to recover the compound from the fermentation broth.
  • the whole fermentation broth is combined with approximately an equal volume of an organic solvent.
  • a water immiscible solvent such as ethyl acetate, methylene chloride, chloroform and the like. Generally several extractions are desirable to achieve maximum recovery.
  • the solvent removes the instant compound as well as other substances lacking the antifungal activity of the instant compound. If the solvent is a water immiscible one, the layers are separated and the organic solvent is evaporated under reduced pressure. The residue is placed onto a chromatography column containing preferably, silica gel.
  • the column retains the desired products and some impurities, but lets many of the impurities, particularly the nonpolar impurities, pass through.
  • the column is washed with a moderately polar organic solvent such as methylene chloride or chloroform to further remove impurities, and is then washed with a mixture of methylene chloride or chloroform and an organic solvent of which acetone, methanol, and ethanol and the like are preferred.
  • the solvent is evaporated and the residue further chromatographed using column chromatography, thin layer chromatography, preparative layer chromatography, high pressure liquid chromatography and the like, with silica gel, aluminum oxide, ion exchange resins, dextran gels and the like, as the chromatographic medium, with various solvents and combinations of solvents as the eluent.
  • Thin layer, high pressure, liquid and preparative layer chromatography may be employed to detect the presence of, and to isolate the instant compound.
  • the use of the foregoing techniques as well as others known to those skilled in the art, will afford purified compositions containing the instant compound.
  • the presence of the desired compounds is determined by analyzing the various chromatographic fractions for physico-chemical characteristics.
  • the structures of the instant compounds have been determined by detailed analysis of the various spectral characteristics of the compounds, in particular their nuclear magnetic resonance, mass, ultraviolet and infrared spectra.
  • the nuclear magnetic resonance (NMR) spectrum for each of Compounds I, II, III and IV are shown in attached FIGS. 1 to 4 respectively.
  • the spectra for Compounds I, III and IV were recorded in CD 3 OD at 60° C. and that for Compound II in CD 3 OD at 25° C.
  • a small amount of ND 4 OD was added to Compound II to effect complete dissolution.
  • Chemical shifts are shown in ppm relative to internal tetramethylsilane (TMS) at 0 ppm.
  • Fields of technology adversely affected by the lack of effective fungicides are many and include the paint, wood, textile, cosmetic, leather, tobacco, fur, rope, paper, pulp, plastics, fuel, rubber and food industries. Fungicides are also utilized for agricultural application, for instance in preventing or minimizing fungus growth on plants, fruits, seeds or soil.
  • the compounds of the invention are effective in controlling the growth of Aspergillus species, for example A. niger, A. flavus, A. fumigatus, A. oryzae, A. luchensis, A. versicolor, A. sydowi, A. nidulans, A. flaucus and A. terreus, Penicillium species, for example, P. notatum, P. roqueforti, P. chrysogenum, P. oxalicum, P. spinulosum, P. martensii, P. citrinium, P. digitatum, P. expansum, P. italicum, P. cyclopium, and P.
  • Aspergillus species for example A. niger, A. flavus, A. fumigatus, A. oryzae, A. luchensis, A. versicolor, A. sydowi, A. nidulans, A. fla
  • fungi may be found on cosmetics, leather, electrical insulation, textiles, and numerous other materials capable of supporting their growth.
  • the compounds of this invention may be employed in treatment of plants, soils, fruits, seeds, fur, wood and the like.
  • the fungicidal effectiveness of these compounds has been demonstrated against soil fungi, such as Rhizoctonia solani, Fusarium solani, and Pythium ultimum, plant fungi, for instance Erysiphe polygoni and Alternaria solani as well as against saprophytes known to attack wood, pulp and lumber such as Lenzites trabea and Ceratocystis pilifera and the fungus Pullularia pullulans which attacks paint.
  • the compounds of this invention are useful in controlling those agricultural fungus infections that attack growing plants.
  • the compounds are particularly effective against those fungi, which cause rice blast, tomato late blight, tomato early blight, wheat leaf rust, bean powdery mildew and tomato Fusarium wilt.
  • the compounds may be utilized in diverse formulations, solid, including finely divided powders and granular materials as well as liquid, such as solutions, emulsions, suspensions, concentrates, emulsifiable concentrate, slurries and the like, depending upon the application intended and the formulation media desired.
  • compositions containing such compounds as essentially active ingredients thereof, which compositions may also include finely divided dry or liquid diluents, extenders, fillers, conditioners and excipients, including various clays, diatomaceous earth, talc, and the like, or water and various organic liquids such as lower alkanols, for example ethanol and isopropanol, or kerosene, benzene, toluene and other petroleum distillate fractions or mixtures thereof.
  • finely divided dry or liquid diluents including various clays, diatomaceous earth, talc, and the like, or water and various organic liquids such as lower alkanols, for example ethanol and isopropanol, or kerosene, benzene, toluene and other petroleum distillate fractions or mixtures thereof.
  • one or more of the compounds may be uniformly distributed in a vehicle that is chemically compatible with the particular compound selected, non-inhibiting with respect to the action of the antifungal and essentially non-injurious to body tissue under the conditions of use.
  • the compounds of the invention may be used in combination one with the other as well as with other fungicidally active materials.
  • 2-(4'-thiazolyl)benzimidazole sorbic acid or its salts, propionic acid or its salts mycostatin
  • soil as used herein is intended to include all media capable of supporting the growth of plants and may include humus, sand, manure, compost, artifically created plant growth solution, and the like.
  • fungicides of the invention are effective against fungal diseases of plants, and may be effectively used either by direct contact with the foliage or systemically, by introduction through the roots.
  • Culture MA 5000 is maintained in the lyophilized state in a 1.0 ml ampoule containing 0.15 ml of a skim milk suspension of the culture.
  • Inoculum Contents of one lyophilization tube into each "B" flask
  • Sterility Streak plates and gram stain
  • Meat Extract 480 gms
  • NZ Amine Type E 800 gms
  • Na 2 HPO 4 30 gms pH to 7.0-7.2
  • Sterility Microscopic examination and YED streak plates at 28° C. and 37° C.
  • Vessel 756-liter fermentor containing 467 liters of medium
  • Sterility Microscopic examination and YED streak plates at 28° C. and 37° C.
  • the second eluate was concentrated in vacuo to 4 liters and eluted to 16 liters with water.
  • This solution was passed through a 16 liter Amberlite XAD-2 column. After a water wash the column was eluted with 48 liters 10% aqueous acetone, 48 liters 20% aqueous acetone and 50% aqueous acetone.
  • the third eluate was concentrated to an aqueous solution of 4 liters which was extracted twice with 8.8 liters butanol. The butanol extracts were concentrated to a small volume and the activity precipitated with the addition of hexane.
  • the precipitate was chromatographed on a 500 ml Mallinkrodt CC-7 silica gel column in acetone with stepwise increases of methanol in acetone. Active cuts were combined and chromatographed on a 225 ml LH-20 column in methanol. Active cuts were combined and rechromatographed on the same column in 25% acetone/methanol yielding 549 mg. Aliquots of this purified preparation were chromatographed on a preparative RP-2, 10 ⁇ m HPLC column (9 mm ⁇ 30 cm) using a methanol - 1% HOAc gradient system. Fractions of the three separated components were combined, concentrated to aqueous solutions and freeze dried to yield the following samples:
  • Example 1 using "E" Stage fermentation 2 was carried out twice and each 400 liter formation was filtered 320 liters through a press after addition of celite. Each mycelial cake was extracted with 320 liters of 50% aqueous acetone for one hour. The acetone extracts were filtered and concentrated to aqueous solutions. These extracts and the filtrates were individually extracted with a half volume of methylene chloride and then each extracted twice with a half volume of butanol. The butanol extracts were combined and evaporated onto 8 liters Grace silica gel. The coated gel was eluted sequentially with ethyl acetate, acetone, 10% methanol/acetone and 100% methanol.
  • the methanol eluate was concentrated (180 gm) onto 700 ml Baker silica gel and placed on top of a two-liter silica gel column. This column was eluted with acetone, then stepwise with 20-40-75% methanol/acetone.
  • the active cuts were concentrated, extracted with butanol (16 gm) and chromatographed on a two-liter EM 25-40 ⁇ m RP C-18 column with isocratic elution of 60/40 methanol/0.01M PO 4 pH 6.5. Cuts were combined based on their zone size and analytical HPLC analysis and rechromatographed on a 200 ml EM 25- 40 ⁇ m RP C-18 column using 55/45 methanol/buffer. Final purification of the individual active components was achieved by chromatography on a 10 ⁇ m RP C-18 column, 9 mm ⁇ 50 cm.

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Abstract

There is disclosed a series of macrolides isolated from the fermentation broth of a microorganism identified as MA-5000 which morphological analysis reveals to be a strain of Streptomyces hygroscopicus. The compounds' structures are present based upon analytical studies. The compounds have antifungal activity.

Description

This is a continuation-in-part of application Ser. No. 791,623, now abandoned, filed Oct. 25, 1985, which is a division of application Ser. No. 593,448, now U.S. Pat. No. 4,575,500, filed Mar. 26, 1984.
SUMMARY OF THE INVENTION
This invention is concerned with novel organic chemical compounds. In particular, it is concerned with phosphate esters which are produced by the fermentation of a nutrient medium with a strain of the microorganism Streptomyces hygroscopicus MA-5000. Thus, it is an object of this invention to provide for such novel compounds, and a method for preparing such products microbiologically. It is a further object of this invention to provide for the recovery and purification of such compounds from the fermentation broth. These substances have antifungal activity, and it is, thus, an additional object of this invention to provide for novel antifungal compositions containing the disclosed compounds. Further objects of this invention will become apparent from the following description of this invention.
DESCRIPTION OF THE INVENTION
In accordance with this invention, there is described a series of novel substances which are prepared by growing under controlled conditions, a previously undescribed strain of a microorganism. The novel compounds are produced by Streptomyces hygroscopicus MA-5000. The compounds are obtained by fermentation and recovered in substantially pure form as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a nuclear magnetic resonance spectrum for Compound I.
FIG. 2 is a nuclear magnetic resonance spectrum for Compound II.
FIG. 3 is a nuclear magnetic resonance spectrum for Compound III.
FIG. 4 is a nuclear magnetic resonance spectrum for Compound IV.
The production and identification of compounds are hereinafter described.
Based on taxonomic studies, the microorganism capable of producing these compounds is of a new strain of the microorganism Streptomyces hygroscopicus. One such culture, isolated from soil, is designated MA-5000 in the culture collection of Merck & Co., Inc., Rahway, N.J. A sample of this culture, capable of producing the herein described compound, has been deposited, without restriction as to availability, in the permanent culture collection of the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Md. 20852, and has been assigned the accession number ATCC 39476.
The morphological and cultural characteristics of Streptomyces hygroscopicus MA-5000 are set forth below:
Cultural Characteristic of:
Streptomyces hygroscopicus MA-5000--ATCC 39476 (V-vegetative growth; A=aerial mycelium; SP=soluble pigment).
Morphology: Sporophores form compact spirals along aerial hyphae. As culture ages, spore chains coalesce to form dark moist areas. Spore surface by electron microscope (TEM) shows a smooth surface on some spores and a rough to warty surface on others.
Oatmeal agar (ISP Medium 3)
V: Reverse--brown edged with dark brown
A: Dark gray mixed with white and light gray, becoming black and moist with age.
SP: Lt. grayish brown.
Czapek Dox agar (sucrose nitrate agar)
V: Reverse--dark brown
A: moderate, grayish white
SP: Lt. brown
Egg albumin agar
V: Flat, spreading, grayish brown
A: Light gray mixed with white and edged with dark gray, some areas becoming moist and black.
SP: Light brown
Glycerol asparagine agar (ISP Medium 5)
V: Reverse--dark yellow brown
A: Light gray mixed with white and yellowish white. Droplets of golden yellow exudate are present.
SP: Lt. tan
Inorganic salts-starch agar (ISP Medium 4)
V: Reverse--light gray-brown edged with dark gray
A: Dark gray mixed with light gray and white, becoming black and moist.
SP: Light grayish-brown
Yeast extract malt extract agar (ISP Medium 2)
V: Reverse--dark yellow brown
A: Dark gray becoming black and moist with age. Droplets of golden yellow exudate also present.
SP: Light brown
Peptone-iron-yeast extract agar
V: tan
A: sparse, whitish
SP: None
Melanin: negative
Nutrient tyrosin agar
V: Rev.--brown
A: Moderate, light gray
SP: Light brown
Tyrosine Agar (ISP Medium 7)
V: Reverse--dark brown
A: Gray edged with dark gray; droplets of golden yellow exudate present
SP: Light brown
Carbon utilization
Pridham-Gottlieb basal medium+1% carbon source;
+=growth; ±=growth poor or questionable;
-=no growth as compared to negative control (no carbon source)
______________________________________                                    
        Glucose +                                                         
        Arabinose                                                         
                ±                                                      
        Cellulose                                                         
                --                                                        
        Fructose                                                          
                +                                                         
        Inositol                                                          
                +                                                         
        Lactose ±                                                      
        Maltose +                                                         
        Mannitol                                                          
                +                                                         
        Mannose +                                                         
        Raffinose                                                         
                ±                                                      
        Rhamnose                                                          
                ±                                                      
        Sucrose +                                                         
        Xylose  ±                                                      
______________________________________
Temperature range (Yeast extract-dextrose+salts agar)
26° C.--Good vegetative growth and sporulation
37° C.--Good vegetative growth scant aerial mycelia
50° C.--No growth
Oxygen requirement (Stab culture in yeast extractdextrose +salts agar)
Aerobic
All readings taken after three weeks at 28° C. unless noted otherwise. pH of all media approximately neutral (6.8-7.2)
A careful comparison of the foregoing data with published descriptions, including Bergey's Manual of Determinative Bacteriology 8th ed (1974); Waksman, The Actinomycetes Vol. II (1961); International Journal of Systematic Bacteriology 18, 68-189, 279-392 (1968); 19, 391-512 (1969); 22, 265-394 (1972); shows a close correlation between the description of a bacterium identified as Streptomyces hygroscopicus and the morphological and cultural characteristics of MA 5000. It is therefore determined that MA-5000 is a new strain of the known species Streptomyces hygroscopicus.
The above description is illustrative of a strain of Streptomyces hygroscopicus MA-5000 which can be employed in the production of the instant compound. However, the present invention also embraces mutants of the above described microorganism. For example, those mutants which are obtained by natural selection or those produced by mutating agents including ionizing radiation such as ultraviolet irradiation, or chemical mutagens such as nitrosoguanidine or the like treatments are also included within the ambit of this invention.
The instant compounds are produced during the aerobic fermentation of suitable aqueous nutrient media under conditions described hereinafter, with a producing strain of Streptomyces hygroscopicus MA-5000. Aqueous media such as those used for the production of many antibiotic substances are suitable for use in this process for the production of this macrocyclic compound.
Such nutrient media contain sources of carbon and nitrogen assimilable by the microorganism and generally low levels of inorganic salts. In addition, the fermentation media may contain traces of metals necessary for the growth of the microorganisms, and production of the desired compound. These are usually present in sufficient concentrations in the complex sources of carbon and nitrogen, which may be used as nutrient sources, but can, of course, be added separately to the medium if desired.
In general, carbohydrates such as sugars, for example dextrose, sucrose, maltose, lactose, dextran, cerelose, corn meal, oat flour, and the like, and starches are suitable sources of assimilable carbon in the nutrient media. The exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between 0.5 and 5% by weight of the medium is satisfactory. These carbon sources can be used individually or several such carbon sources may be combined in the same medium.
Various nitrogen sources such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, corn meal, oat flour, soybean meal, caesin hydrolysates, yeast extracts, corn steep liquors, distillers solubles, cottonseed meal, meat extract and the like, are readily assimilable by Streptomyces hygroscopicus MA 5000 in the production of the instant compound. The various sources of nitrogen can be used alone or in combination in amounts ranging from 0.2 to 6% by weight of the medium.
Among the nutrient inorganic salts, which can be incorporated in the culture media are the customary salts capable of yielding sodium, potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride, carbonate, and like ions. Also included are trace metals such as cobalt, manganese, and the like. For the best production of the instant compound, the addition of calcium carbonate to the production medium is most preferred.
It should be noted that the media described hereinbelow and in the Examples are merely illustrative of the wide variety of media, which may be employed, and are not intended to be limitative.
The following are Examples of media suitable for growing strains of Streptomyces hygroscopicus MA-5000.
______________________________________                                    
Medium A                                                                  
Dextrose              1.0       g.                                        
Soluble starch        10.0      g.                                        
Beef extract          3.0       g.                                        
Yeast autolysate (As ardamine                                             
                      5.0       g.                                        
PH available from Yeast                                                   
Products Inc., Clifton, N.J.)                                             
NZ Amine-E (caesin hydrolysate-                                           
                      5.0       g.                                        
available from Humko-Sheffield-                                           
Memphis, Tenn.)                                                           
MgSO.sub.4.7H.sub.2 O 0.05      g.                                        
Phosphate Buffer      2.0       ml.                                       
CaCO.sub.3            0.5       g.                                        
Distilled water       1000      ml.                                       
pH                    7.0-7.2                                             
Phosphate Buffer                                                          
KH.sub.2 PO.sub.4     91.0      g.                                        
Na.sub.2 HPO.sub.4    95.0      g.                                        
Distilled water       1000      ml.                                       
pH                    7.0                                                 
Medium B                                                                  
Tomato paste          20.0      g.                                        
Primary yeast         10.0      g.                                        
Dextrin (CPC starch)  20.0      g.                                        
CoCl.sub.2.6H.sub.2 O 0.005     g.                                        
Distilled water       1000      ml.                                       
pH                    7.2-7.4                                             
Medium C                                                                  
Corn meal             20.0      g.                                        
Distillers solubles   10.0      g.                                        
Soybean meal          15.0      g.                                        
Sodium citrate        4.0       g.                                        
CaCl.sub.2.2H.sub.2 O 0.5       g.                                        
MgSO.sub.4.7H.sub.2 O 0.1       g.                                        
CoCl.sub.2.6H.sub.2 O 0.01      g.                                        
FeSO.sub.4.2H.sub.2 O 0.01      g.                                        
Polyglycol P2000 (Polypropylene                                           
                      2.5       mg.                                       
glycol mw 2000)                                                           
Distilled water       1000      ml.                                       
pH                    6.5                                                 
Medium D                                                                  
LACTOSE               20.0      g.                                        
Distillers solubles   15.0      g.                                        
Autolyzed yeast (Ardamine PH)                                             
                      5.0       g.                                        
Distilled water       q.s. to 1000                                        
                                ml.                                       
pH                    7.0                                                 
Medium E                                                                  
Tomato paste          40.0      g.                                        
Oat flour             10.0      g.                                        
Distilled water       1000      ml.                                       
pH                    7.0                                                 
Medium F                                                                  
Corn Steep Liquor     15.0      g.                                        
(NH.sub.4).sub.2 SO.sub.4                                                 
                      4.0       g.                                        
CaCO.sub.3            6.0       g.                                        
Soluble Starch        20.0      g.                                        
Corn meal             1.0       g.                                        
Soybean meal          4.0       g.                                        
Glucose               5.0       g.                                        
KH.sub.2 PO.sub.4     0.3       g.                                        
Lard oil              2.5       g.                                        
Distilled water       1000      ml.                                       
pH                    6.7                                                 
Medium G                                                                  
Dextrose              10.0      g.                                        
Asparagine            1.0       g.                                        
K.sub.2 HPO.sub.4     0.1       g.                                        
MgSO.sub.4.7H.sub.2 O 0.5       g.                                        
Yeast Extract         0.5       g.                                        
Oat Flour             10.0      g.                                        
CaCO.sub.3            3.0       g.                                        
Trace Element Mix     10.0      ml.                                       
Distilled water       1000      ml.                                       
Adjust pH to          7.2                                                 
Trace Element Mix                                                         
FeSO.sub.4.7H.sub.2 O 1000      mg.                                       
MnSO.sub.4.4H.sub.2 O 1000      mg.                                       
CuCl.sub.2.2H.sub.2 O 25        mg.                                       
CaCl.sub.2.2H.sub.2 O 100       mg.                                       
H.sub.3 BO.sub.3      56        mg.                                       
(NH.sub.4).sub.6 MO.sub.4 O.sub.24.6H.sub.2 O                             
                      19        mg.                                       
ZnSO.sub.4.7H.sub.2 O 200       mg.                                       
Distilled water       1000      ml.                                       
Medium H                                                                  
Medium G              1000      ml.                                       
Oat Flour             10        g.                                        
pH                    7.2                                                 
______________________________________
The fermentation employing Streptomyces hygroscopicus MA 5000 can be conducted at temperatures ranging from about 20° C. to about 40° C. For optimum results, it is most convenient to conduct these fermentations at a temperature in the range of from about 24° C. to about 30° C. Temperatures of about 27°-28° C. are most preferred. The pH of the nutrient medium suitable for producing the instant compounds can vary from about 5.0 to 8.5 with a preferred range of from about 6.0 to 7.5.
Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in a flask employing known sterile techniques, inoculating the flask with either spores or vegetative cellular growth of Streptomyces hygroscopicus MA-5000 loosely stoppering the flask with cotton and permitting the fermentation to proceed in a constant room temperature of about 28° C. on a rotary shaker at from 95 to 300 rpm for about 2 to 10 days. For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium. The nutrient medium is made up in the tank and after sterilization is inoculated with a source of vegetative cellular growth of Streptomyces hygroscopicus MA 5000. The fermentation is allowed to continue for from 1 to 8 days while agitating and/or aerating the nutrient medium at a temperature in the range of from about 24° to 37° C. The degree of aeration is dependent upon several factors such as the size of the fermenter, agitation speed, and the like. Generally the larger scale fermentations are agitated at about 95 to 300 RPM and about 2 to 20 cubic feet per minute (CFM) of air.
The novel compound of this invention is found primarily in the mycelium on termination of the Streptomyces hygroscopicus MA-5000 fermentation and may be removed and separated therefrom as described below.
The separation of the novel compound from the whole fermentation broth and the recovery of said compounds is carried out by solvent extraction and application of chromatographic fractionations with various chromatographic techniques and solvent systems.
The instant compound has slight solubility in water, but is soluble in organic solvents. This property may be conveniently employed to recover the compound from the fermentation broth. Thus, in one recovery method, the whole fermentation broth is combined with approximately an equal volume of an organic solvent. While any organic solvent may be employed, it is preferable to use a water immiscible solvent such as ethyl acetate, methylene chloride, chloroform and the like. Generally several extractions are desirable to achieve maximum recovery. The solvent removes the instant compound as well as other substances lacking the antifungal activity of the instant compound. If the solvent is a water immiscible one, the layers are separated and the organic solvent is evaporated under reduced pressure. The residue is placed onto a chromatography column containing preferably, silica gel. The column retains the desired products and some impurities, but lets many of the impurities, particularly the nonpolar impurities, pass through. The column is washed with a moderately polar organic solvent such as methylene chloride or chloroform to further remove impurities, and is then washed with a mixture of methylene chloride or chloroform and an organic solvent of which acetone, methanol, and ethanol and the like are preferred. The solvent is evaporated and the residue further chromatographed using column chromatography, thin layer chromatography, preparative layer chromatography, high pressure liquid chromatography and the like, with silica gel, aluminum oxide, ion exchange resins, dextran gels and the like, as the chromatographic medium, with various solvents and combinations of solvents as the eluent. Thin layer, high pressure, liquid and preparative layer chromatography may be employed to detect the presence of, and to isolate the instant compound. The use of the foregoing techniques as well as others known to those skilled in the art, will afford purified compositions containing the instant compound. The presence of the desired compounds is determined by analyzing the various chromatographic fractions for physico-chemical characteristics. The structures of the instant compounds have been determined by detailed analysis of the various spectral characteristics of the compounds, in particular their nuclear magnetic resonance, mass, ultraviolet and infrared spectra.
The mass spectral data were obtained on the trimethylsilyl derivatives of the instant compounds, prepared by treatment with bis(trimethylsilyl)trifluoroacetamide (BSTFA), 10:1 (v/v) in pyridine under nitrogen at room temperature for 18 hours to afford molecular ions in their electron impact mass spectra. Analogous silylation with perdeutero-BSTFA allowed unambiguous determination of the number of silyl groups in each molecule. High resolution exact mass measurements indicated the following molecular formulae. (The underivatized nominal molecular weights and molecular formulae have been included in the table for clarity.)
__________________________________________________________________________
Com-                                                                      
    Neat                                                                  
        Molecular                                                         
               Corresponding                                              
                       Silyl                                              
                            Silyl                                         
pound                                                                     
    Weight                                                                
        Formula                                                           
               Calculated                                                 
                       Found                                              
                            Derivative                                    
__________________________________________________________________________
I   599 C.sub.29 H.sub.46 NO.sub.10 P                                     
               959.4836                                                   
                       959.4847                                           
                            C.sub.29 H.sub.46 NO.sub.10 P + (C.sub.3      
                            H.sub.8 Si).sub.5                             
II  513 C.sub.25 H.sub.40 NO.sub.8 P                                      
               873.4468                                                   
                       873.4452                                           
                            C.sub.25 H.sub.40 NO.sub.8 P + (C.sub.3       
                            H.sub.8 Si).sub.5                             
III 613 C.sub.30 H.sub.48 NO.sub.10 P                                     
               973.4992                                                   
                       973.4993                                           
                            C.sub.38 H.sub.48 NO.sub.10 P + (C.sub.3      
                            H.sub.8 Si).sub.5                             
V   614 C.sub.30 H.sub.47 O.sub.11 P                                      
               974.4832                                                   
                       974.4766                                           
                            C.sub.30 H.sub.47 O.sub.11 P + (C.sub.3       
                            H.sub.8 Si).sub.5                             
__________________________________________________________________________
The nuclear magnetic resonance (NMR) spectrum for each of Compounds I, II, III and IV are shown in attached FIGS. 1 to 4 respectively. The spectra for Compounds I, III and IV were recorded in CD3 OD at 60° C. and that for Compound II in CD3 OD at 25° C. A small amount of ND4 OD was added to Compound II to effect complete dissolution. Chemical shifts are shown in ppm relative to internal tetramethylsilane (TMS) at 0 ppm.
Based on these experimental data, the instant compounds are believed to have the following structural formula: ##STR1## wherein the compounds have the following values for R1, R2 and R3 :
______________________________________                                    
R.sub.1           R.sub.2   R.sub.3                                       
______________________________________                                    
       ##STR2##       NH.sub.3.sup.⊕                                  
                                OPO.sub.3 H.sup.⊖                 
II    H               NH.sub.3.sup.⊕                                  
                                OPO.sub.3 H.sup.⊖                 
III                                                                       
       ##STR3##       NH.sub.3.sup.⊕                                  
                                OPO.sub.3 H.sup.⊖                 
IV                                                                        
       ##STR4##       OH        OPO.sub.3 H.sub.2                         
______________________________________
Fields of technology adversely affected by the lack of effective fungicides are many and include the paint, wood, textile, cosmetic, leather, tobacco, fur, rope, paper, pulp, plastics, fuel, rubber and food industries. Fungicides are also utilized for agricultural application, for instance in preventing or minimizing fungus growth on plants, fruits, seeds or soil.
Although many antifungal agents have been described and used heretofore in an effort to control fungi, none are entirely satisfactory and continued losses resulting from fungal attack make the problem of control a serious and lasting one. The number of fungicides practically useful in combatting fungal growth have been small and only in a few cases have synthetic organic chemicals been found applicable.
The compounds of the invention are effective in controlling the growth of Aspergillus species, for example A. niger, A. flavus, A. fumigatus, A. oryzae, A. luchensis, A. versicolor, A. sydowi, A. nidulans, A. flaucus and A. terreus, Penicillium species, for example, P. notatum, P. roqueforti, P. chrysogenum, P. oxalicum, P. spinulosum, P. martensii, P. citrinium, P. digitatum, P. expansum, P. italicum, P. cyclopium, and P. funiculosum, Neurospora series such as N. sitophila, Phoma species such as P. terrestrius, Rhizopus species, Alternaria species such as A. solani, Chaetomium species such as C. globosum, Chaetomicum species, for example, C. clivaceum, and Monilia species such as M. sitophila and M. nigra. The above fungi may be found on cosmetics, leather, electrical insulation, textiles, and numerous other materials capable of supporting their growth.
The compounds of this invention may be employed in treatment of plants, soils, fruits, seeds, fur, wood and the like. The fungicidal effectiveness of these compounds has been demonstrated against soil fungi, such as Rhizoctonia solani, Fusarium solani, and Pythium ultimum, plant fungi, for instance Erysiphe polygoni and Alternaria solani as well as against saprophytes known to attack wood, pulp and lumber such as Lenzites trabea and Ceratocystis pilifera and the fungus Pullularia pullulans which attacks paint.
In particular the compounds of this invention are useful in controlling those agricultural fungus infections that attack growing plants. The compounds are particularly effective against those fungi, which cause rice blast, tomato late blight, tomato early blight, wheat leaf rust, bean powdery mildew and tomato Fusarium wilt.
It should be understood that the compounds may be utilized in diverse formulations, solid, including finely divided powders and granular materials as well as liquid, such as solutions, emulsions, suspensions, concentrates, emulsifiable concentrate, slurries and the like, depending upon the application intended and the formulation media desired.
Thus it will be appreciated that compounds of this invention may be employed to form fungicidally active compositions containing such compounds as essentially active ingredients thereof, which compositions may also include finely divided dry or liquid diluents, extenders, fillers, conditioners and excipients, including various clays, diatomaceous earth, talc, and the like, or water and various organic liquids such as lower alkanols, for example ethanol and isopropanol, or kerosene, benzene, toluene and other petroleum distillate fractions or mixtures thereof.
When the active agents are employed in preventing topical fungal growth, one or more of the compounds may be uniformly distributed in a vehicle that is chemically compatible with the particular compound selected, non-inhibiting with respect to the action of the antifungal and essentially non-injurious to body tissue under the conditions of use.
It should be understood that the compounds of the invention may be used in combination one with the other as well as with other fungicidally active materials. For instance, a mixture of the active compounds with 2-(4'-thiazolyl)benzimidazole sorbic acid or its salts, propionic acid or its salts, mycostatin, sodium diacetate, trichomycin, amphotericin, griseofulvin, undecylenic acid, chlorquinadol, 5,7-dichloro-8-hydroxyquinoline (Vioform), sodium o-phenylphenate, o-phenylphenol, biphenyl, chlorinated phenols, sodium benzoate, dehydroacetic acid and its salts or esters of parahydroxybenzoic acid, such as the methyl and propyl ester (parabens) can be used to give fungicidal effect when used in appropriate concentrations. It is quite clear, too, that the compounds defined above may be used in conjunction with effective antibacterial materials in appropriate instances so as to combine the action of each in such a situation as to be particularly useful, for instance, in applications where the presence of bacteria creates undesirable results alongside the detrimental action of fungi.
It has been found that growth of various fungi existing in soil is limited or terminated by the addition to the soil of minor quantities of the compounds described. The term soil as used herein is intended to include all media capable of supporting the growth of plants and may include humus, sand, manure, compost, artifically created plant growth solution, and the like.
We have also found that the fungicides of the invention are effective against fungal diseases of plants, and may be effectively used either by direct contact with the foliage or systemically, by introduction through the roots.
The following examples are being provided in order that the instant invention may be more fully understood. Such examples are not to be construed as being limitative of the invention.
EXAMPLE 1
1. "A" Stage:
Culture MA 5000 is maintained in the lyophilized state in a 1.0 ml ampoule containing 0.15 ml of a skim milk suspension of the culture.
2. "B" Stage:
Vessel: 250 ml 3 baffled Erlenmeyer flask containing 50 ml medium per flask.
______________________________________                                    
Medium:               Wt/Vol                                              
______________________________________                                    
Dextrose              0.1%                                                
Soluble Starch        1.0%                                                
Beef Extract          0.3%                                                
Ardamine PH           0.5%                                                
NZ-Amine Type E       0.5%                                                
MgSO.sub.4.7H.sub.2 O 0.005%                                              
1.34 M Phosphate Buffer                                                   
                      0.02% vol/vol                                       
CaCO.sub.3 (after pH adjustment)                                          
                      0.05%                                               
______________________________________
PHOSPHATE BUFFER
KH2 PO4 9.1%
Na2 HPO4 9.5%
Inoculum: Contents of one lyophilization tube into each "B" flask
Incubation: 48 hours at 28° C. on a rotary shaker with a 2" throw rotating at 220 RPM.
Sterility: Streak plates and gram stain
3. "C" Stage:
Vessel: 2-liter 3 baffled Erlenmeyer flask containing 500 ml medium
Medium: Same as "B" Stage
Inoculum: 10 ml from "B" Stage
Incubation: Same as "B" Stage
Sterility: Same as "B" Stage
4. "D" Stage:
Vessel: 189-liter stainless steel fermentor containing 160 liters of medium
Medium:
Dextrose (Cerelose): 160 gms
Starch Modified CPC: 1600 gms
Meat Extract: 480 gms
Ardamine pH: 800 gms
NZ Amine Type E: 800 gms
MgSO4 ·7 H2 O: 8 gms
KH2 PO4 : 29 gms
Na2 HPO4 : 30 gms pH to 7.0-7.2
Add CaCO3 : 80 gms
Sterilization: 121° C. for 15 minutes
Inoculum: 500 ml from "C" Stage
Incubation:
a) Temp: 28° C.
b) Airflow: 3 ft3 /min
c) Agitation: 150 RPM
d) Pressure: 30 psig
e) Cycle: 48 hrs
Defoamer: Polyglycol 2000
Sterility: Microscopic examination and YED streak plates at 28° C. and 37° C.
5. "E" Stage:
Vessel: 756-liter fermentor containing 467 liters of medium
______________________________________                                    
Medium:       Fermentation 1                                              
                          Fermentation 2                                  
______________________________________                                    
Corn Meal     9340    gms     9680   gms                                  
Distillers Solubles                                                       
              4670    gms     4840   gms                                  
Soybean Meal  7005    gms     7260   gms                                  
CaCl.sub.2.2H.sub.2 O                                                     
              233.5   gms     242    gms                                  
MgSO.sub.4.7H.sub.2 O                                                     
              46.7    gms     48.4   gms                                  
CoCl.sub.2.6H.sub.2 O                                                     
              4.7     gms     4.8    gms                                  
FeSO.sub.4.7H.sub.2 O                                                     
              4.7     gms     4.8    gms                                  
CaCO.sub.3    1868    gms     1936   gms                                  
Polyglycol P-2000                                                         
              116     ml      1.2    l                                    
pH to         6.5             6.5                                         
Volume        467     liters  484    liters                               
______________________________________
Sterilization: 121° C. for 15 minutes
Inoculum: 43 liters from "D" Stage
Incubation:
a) Temp: 28° C.
b) Airflow: 10 ft3 /min
c) Agitation: 130 RPM
d) Pressure: 13 psig
e) Cycle: 3 days
Defoamer: Polyglycol P-2000
Sterility: Microscopic examination and YED streak plates at 28° C. and 37° C.
Harvest: Into drums for work-up as in Examples 2 and 3
EXAMPLE 2
Four hundred liters of whole broth from Example 1 "E" Stage fermentation 1, were filtered through a press after the addition of celite, a filter aid. The mycelial cake was extracted with 32 liters of 50% aqueous acetone for one hour. This slurry was filtered and the extract concentrated in vacuo to an aqueous solution. This concentrate was added to the filtered broth and the entire solution was extracted twice with a half volume of methylene chloride. The aqueous phase was then passed through 40 liters Dowex 1X2 (C1-) column. After a water wash the column was eluted with 120 liters of 5% sodium chloride followed by 120 liters of 3% ammonium chloride in 90% methanol. The second eluate was concentrated in vacuo to 4 liters and eluted to 16 liters with water. This solution was passed through a 16 liter Amberlite XAD-2 column. After a water wash the column was eluted with 48 liters 10% aqueous acetone, 48 liters 20% aqueous acetone and 50% aqueous acetone. The third eluate was concentrated to an aqueous solution of 4 liters which was extracted twice with 8.8 liters butanol. The butanol extracts were concentrated to a small volume and the activity precipitated with the addition of hexane. The precipitate was chromatographed on a 500 ml Mallinkrodt CC-7 silica gel column in acetone with stepwise increases of methanol in acetone. Active cuts were combined and chromatographed on a 225 ml LH-20 column in methanol. Active cuts were combined and rechromatographed on the same column in 25% acetone/methanol yielding 549 mg. Aliquots of this purified preparation were chromatographed on a preparative RP-2, 10 μm HPLC column (9 mm×30 cm) using a methanol - 1% HOAc gradient system. Fractions of the three separated components were combined, concentrated to aqueous solutions and freeze dried to yield the following samples:
Component I: 18 mg
Component II: 16 mg
Component III: 14 mg
The remainder of the LH-20 rich cut was chromatographed on a preparative RP-18, 10 μm HPLC column (9 mm×30 cm) using the isocratic system 500/450/50 methanol/water/1.0M PO4 pH 6.5 to yield the following samples:
Component I: 15.8 mg
Component II: 9.9 mg
EXAMPLE 2
The fermentation of Example 1 using "E" Stage fermentation 2 was carried out twice and each 400 liter formation was filtered 320 liters through a press after addition of celite. Each mycelial cake was extracted with 320 liters of 50% aqueous acetone for one hour. The acetone extracts were filtered and concentrated to aqueous solutions. These extracts and the filtrates were individually extracted with a half volume of methylene chloride and then each extracted twice with a half volume of butanol. The butanol extracts were combined and evaporated onto 8 liters Grace silica gel. The coated gel was eluted sequentially with ethyl acetate, acetone, 10% methanol/acetone and 100% methanol. The methanol eluate was concentrated (180 gm) onto 700 ml Baker silica gel and placed on top of a two-liter silica gel column. This column was eluted with acetone, then stepwise with 20-40-75% methanol/acetone. The active cuts were concentrated, extracted with butanol (16 gm) and chromatographed on a two-liter EM 25-40 μm RP C-18 column with isocratic elution of 60/40 methanol/0.01M PO4 pH 6.5. Cuts were combined based on their zone size and analytical HPLC analysis and rechromatographed on a 200 ml EM 25- 40 μm RP C-18 column using 55/45 methanol/buffer. Final purification of the individual active components was achieved by chromatography on a 10 μm RP C-18 column, 9 mm×50 cm.
Component II: 40 mg
Component III: 6.7 mg
Component IV: 2.5 mg

Claims (1)

What is claimed is:
1. A process for preparing a compound having the formula: ##STR5## wherein (1) when R1 is ##STR6## R2 is --NH3.sup.⊕, and R3 is --O--PO3 H,.sup.⊖ the compound has a molecular weight of 599, a molecular formula of C29 H46 NO10 P and an NMR spectrum substantially as shown in FIG. 1;
(2) when R1 is H, R2 is --NH3.sup.⊕, and R3 is --O--PO3 H.sup.⊖, the compound has a molecular weight of 513, a molecular formula of C25 H40 NO8 P and an NMR spectrum substantially as shown in FIG. 2;
(3) when R1 ##STR7## R2 is --NH3.sup.⊕, and R3 is --O--PO3 H.sup.⊖, the compound has a molecular weight of 613, a molecular formula of C30 H48 NO10 P, and an NMR spectrum substantially as shown in FIG. 3;
(4) when R1 is ##STR8## R2 is OH, and R3 is --O--PO3 H2, the compound has a molecular weight of 614, a molecular formula of C30 H47 O11 P, and an NMR spectrum substantially as shown in FIG. 4; or a mixture of said compounds,
which comprises
(a) fermenting the strain Streptomyces hygroscopicus MA 5000, ATCC 39476, in an aqueous nutrient medium containing an assimilable source of carbon, an assimilable source of nitrogen, and inorganic salts, and
(b) isolating the compound from the fermentation medium by solvent extraction and chromatographic fractionation.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994008933A1 (en) * 1992-10-19 1994-04-28 Merck & Co., Inc. Antibiotic agents

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3068706B2 (en) * 1992-02-26 2000-07-24 塩野義製薬株式会社 Aldose reductase inhibitor
JP3124373B2 (en) * 1992-03-13 2001-01-15 塩野義製薬株式会社 Immunosuppressant
DE19536328A1 (en) * 1995-09-29 1997-04-03 Remmers Bauchemie Gmbh Agent for protecting technical materials against damage and destruction by harmful organisms based on natural active ingredients

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3094460A (en) * 1959-01-20 1963-06-18 Upjohn Co Decoyinine
GB1356080A (en) * 1972-12-28 1974-06-12 Shionogi & Co Antibiotic a-103-a and process for the production thereof
US4269971A (en) * 1978-12-29 1981-05-26 Taisho Pharmaceutical Co., Ltd. Antibiotic TM-531
CA1151091A (en) * 1980-07-17 1983-08-02 Tsutomu Nishida Oa-7653 substance

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3094460A (en) * 1959-01-20 1963-06-18 Upjohn Co Decoyinine
GB1356080A (en) * 1972-12-28 1974-06-12 Shionogi & Co Antibiotic a-103-a and process for the production thereof
US4269971A (en) * 1978-12-29 1981-05-26 Taisho Pharmaceutical Co., Ltd. Antibiotic TM-531
CA1151091A (en) * 1980-07-17 1983-08-02 Tsutomu Nishida Oa-7653 substance

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Wallington et al., In Progress in Industrial Microbiology, 1983, pp. 7 36. *
Wallington et al., In Progress in Industrial Microbiology, 1983, pp. 7-36.

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994008933A1 (en) * 1992-10-19 1994-04-28 Merck & Co., Inc. Antibiotic agents

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